Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells lyse virus-infected cells and possibly emerging tumor cells but they also contribute to transplant rejection and perhaps to autoimmune tissue destruction. One of the lytic molecules of CTL and NK cells is a pore-forming protein known as perforin. Perforin gene expression is restricted to CTL and NK cells. While NK cells express perforin constitutively, CTL express perforin upon their activation. Our objective is to characterize relevant molecular mechanisms 1) that determine the CTL and NK-cell restricted expression of perforin; 2) that determine the induction of perforin in CTL upon their activation; and we propose 3) to identify the transcription factors involved. Within the first objective, we will characterize a cell-type specific cis-acting element in the 5' flanking region of the perforin gene (specific aim 1. 1), determine the role of the proximal perforin promoter (specific aim 1.2) and analyze and determine the role of the chromatin structure of the perforin gene locus (specific aim 1.3). To achieve these aims we will analyze fragments of the perforin 5' flanking and promoter region for their control over the "CAT" reporter gene after transient transfection into different cell types and will compare DNase I hypersensitivity sites of the perforin gene locus between different cell types. Within the second objective, we will determine whether perforin mRNA induction in CTL occurs primarily at the transcriptional level or the posttranscriptional level (specific aim 2.1). We anticipate that the regulation event takes place at the transcriptional level based on our preliminary data and therefore propose to identify regulatory elements involved (specific aim 2.2). To achieve these aims we will analyze, by Northern blot analysis and nuclear run-off assay, perforin gene induction or upregulation in cloned CTL in response to IL-2, T-cell receptor signals or second-messenger pathway activating agents. We will identify the cis-acting responsive regions by reporter gene analysis as outlined above. Within the third objective, we propose to identify putative transcription factors acting on relevant regulatory elements defined in the previous aims (specific aim 3). We will apply in vitro DNase I footprinting and will analyze a particular DNA binding site by gel shift analysis.